Sphingosine-1-phosphate promotes osteogenesis by stimulating osteoblast growth and neovascularization in a vascular endothelial growth factor-dependent manner.

Sphingosine-1-phosphate (S1P) plays multiple roles in bone metabolism and regeneration. Here, we have identified a novel S1P-regulated osteoanabolic mechanism functionally connecting osteoblasts (OBs) to the highly specialized bone vasculature. We demonstrate that S1P/S1PR3 signaling in OBs stimulates vascular endothelial growth factor a (VEGFa) expression and secretion to promote bone growth in an autocrine and boost osteogenic H-type differentiation of bone marrow endothelial cells in a paracrine manner. VEGFa-neutralizing antibodies and VEGF receptor inhibition by axitinib abrogated OB growth in vitro and bone formation in male C57BL/6J in vivo following S1P stimulation and S1P lyase inhibition, respectively. Pharmacological S1PR3 inhibition and genetic S1PR3 deficiency suppressed VEGFa production, OB growth in vitro, and inhibited H-type angiogenesis and bone growth in male mice in vivo. Together with previous work on the osteoanabolic functions of S1PR2 and S1PR3, our data suggest that S1P-dependent bone regeneration employs several nonredundant positive feedback loops between OBs and the bone vasculature. The identification of this yet unappreciated aspect of osteoanabolic S1P signaling may have implications for regular bone homeostasis as well as diseases where the bone microvasculature is affected such as age-related osteopenia and posttraumatic bone regeneration.

Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates bone growth and regeneration. In the present study, a novel regenerative mechanism was connected to S1P signaling within the bone. Activation of its receptor S1PR3 in bone-forming osteoblasts led to secretion of vascular endothelial growth factor a (VEGFa), the most potent vessel-stimulating factor. This stimulated the development of specialized vessels of the bone marrow, the H-type vessels, that supported overall bone regeneration. These findings foster our understanding of regular bone metabolism and suggest that S1P-based drugs may help treat diseases such as age-related osteopenia and posttraumatic bone regeneration, conditions crucially dependent on functional bone microvasculature.

Agonist-induced activation of the S1P receptor 2 constitutes a novel osteoanabolic therapy for the treatment of osteoporosis in mice.

BACKGROUND AND PURPOSE: Osteoporosis is a worldwide epidemic but pharmacological agents to stimulate new bone formation are scarce. We have shown that increasing tissue levels of sphingosine-1-phosphate (S1P) by blocking its degradation by the S1P lyase has pronounced osteoanabolic effect in mouse osteoporosis models by stimulating osteoblast differentiation through the S1P receptor 2 (S1P2). However, S1P lyase inhibitors have side effects complicating potential clinical use. Here, we tested whether direct S1P2 engagement by the S1P2 agonist CYM5520 exerted osteoanabolic potential in estrogen deficiency-induced osteopenia in mice. We compared its efficacy to LX2931, a novel S1P lyase inhibitor currently tested in rheumatoid arthritis. EXPERIMENTAL APPROACH: CYM5520, LX2931 or vehicle were administered to ovariectomized mice for 6 weeks beginning 5 weeks after ovariectomy, Bone mass, cellular composition and mechanical strength were assessed by microCT, histomorphometry and three point bending tests. Plasma markers of bone metabolism were analyzed by ELISA. KEY RESULTS: Therapeutic treatment with CYM5520 and LX2931 clearly increased long bone and vertebral bone mass to impressive 3-5 fold over vehicle in osteopenic ovariectomized mice. As expected, lymphopenia was a side effect of LX2931, whereas none occurred with CYM5520. Consistent with an osteoanabolic effect, CYM5520 increased osteoblast number, osteoid surface and alkaline phosphatase area 2-3 fold over vehicle. Plasma concentrations of the osteoanabolic marker procollagen I C-terminal propeptide were also elevated by CYM5520 and LX2931. LX2931 but not yet CYM5520 increased cortical thickness and mechanical strength without affecting mineral density. CONCLUSION AND IMPLICATIONS: Treatment with a pharmacological S1P2 agonist corrected ovariectomy-induced osteopenia in mice by inducing new bone formation thus constituting a novel osteoanabolic approach to osteoporosis.

Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss.

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2 potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38-GSK3ß-ß-catenin and WNT5A-LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

Hepatocyte Nuclear Factor 1A Is a Cell-Intrinsic Transcription Factor Required for B Cell Differentiation and Development in Mice.

The hepatocyte NF (HNF) family of transcription factors regulates the complex gene networks involved in lipid, carbohydrate, and protein metabolism. In humans, HNF1A mutations cause maturity onset of diabetes in the young type 3, whereas murine HNF6 participates in fetal liver B lymphopoiesis. In this study, we have identified a crucial role for the prototypical member of the family HNF1A in adult bone marrow B lymphopoiesis. HNF1A(-/-) mice exhibited a clear reduction in total blood and splenic B cells and a further pronounced one in transitional B cells. In HNF1A(-/-) bone marrow, all B cell progenitors-from pre-pro-/early pro-B cells to immature B cells-were dramatically reduced and their proliferation rate suppressed. IL-7 administration in vivo failed to boost B cell development in HNF1A(-/-) mice, whereas IL-7 stimulation of HNF1A(-/-) B cell progenitors in vitro revealed a marked impairment in STAT5 phosphorylation. The B cell differentiation potential of HNF1A(-/-) common lymphoid progenitors was severely impaired in vitro, and the expression of the B lymphopoiesis-promoting transcription factors E2A, EBF1, Pax5, and Bach2 was reduced in B cell progenitors in vivo. HNF1A(-/-) bone marrow chimera featured a dramatic defect in B lymphopoiesis recapitulating that of global HNF1A deficiency. The HNF1A(-/-) lymphopoiesis defect was confined to B cells as T lymphopoiesis was unaffected, and bone marrow common lymphoid progenitors and hematopoietic stem cells were even increased. Our data demonstrate that HNF1A is an important cell-intrinsic transcription factor in adult B lymphopoiesis and suggest the IL-7R/STAT5 module to be causally involved in mediating its function.

Sphingosine-1-phosphate receptor 3 promotes recruitment of monocyte/macrophages in inflammation and atherosclerosis.

RATIONALE: The role of sphingosine-1-phosphate (S1P) and its receptors in the pathogenesis of atherosclerosis has not been investigated. OBJECTIVE: We hypothesized that the S1P receptor 3 (S1P(3)) plays a causal role in the pathogenesis of atherosclerosis. METHODS AND RESULTS: We examined atherosclerotic lesion development in mice deficient for S1P(3) and apolipoprotein (Apo)E. Although S1P(3) deficiency did not affect lesion size after 25 or 45 weeks of normal chow diet, it resulted in a dramatic reduction of the monocyte/macrophage content in lesions of S1P(3)(-/-)/ApoE(-/-) double knockout mice. To search for putative defects in monocyte/macrophage recruitment, we examined macrophage-driven inflammation during thioglycollate-induced peritonitis. Elicited peritoneal macrophages were reduced in S1P(3)-deficient mice and expressed lower levels of tumor necrosis factor-α and monocyte chemoattractant protein-1. Bone marrow-derived S1P(3)-deficient macrophages produced less MCP-1 in response to lipopolysaccharide stimulation. In vitro, S1P was chemotactic for wild-type but not S1P(3)-deficient peritoneal macrophages. In vivo, S1P concentration increased rapidly in the peritoneal cavity after initiation of peritonitis. Treatment with the S1P analog FTY720 attenuated macrophage recruitment to the peritoneum. Studies in bone marrow chimeras showed that S1P(3) in both hematopoietic and nonhematopoietic cells contributed to monocyte/macrophage accumulation in atherosclerotic lesions. Finally, S1P(3) deficiency increased the smooth muscle cell content of atherosclerotic lesions and enhanced neointima formation after carotid ligation arguing for an antiproliferative/antimigratory role of S1P(3) in the arterial injury response. CONCLUSIONS: Our data suggest that S1P(3) mediates the chemotactic effect of S1P in macrophages in vitro and in vivo and plays a causal role in atherosclerosis by promoting inflammatory monocyte/macrophage recruitment and altering smooth muscle cell behavior.

Survivin determines cardiac function by controlling total cardiomyocyte number.

BACKGROUND: Survivin inhibits apoptosis and regulates cell division in many organs, but its function in the heart is unknown. METHODS AND RESULTS: We show that cardiac-specific deletion of survivin resulted in premature cardiac death. The underlying cause was a dramatic reduction in total cardiomyocyte numbers as determined by a stereological method for quantification of cells per organ. The resulting increased hemodynamic load per cell led to progressive heart failure as assessed by echocardiography, magnetic resonance imaging, positron emission tomography, and invasive catheterization. The reduction in total cardiomyocyte number in alpha-myosin heavy chain (MHC)-survivin(-/-) mice was due to an approximately 50% lower mitotic rate without increased apoptosis. This occurred at the expense of DNA accumulation because survivin-deficient cardiomyocytes displayed marked DNA polyploidy indicative of consecutive rounds of DNA replication without cell division. Survivin small interfering RNA knockdown in neonatal rat cardiomyocytes also led to polyploidization and cell cycle arrest without apoptosis. Adenoviral overexpression of survivin in cardiomyocytes inhibited doxorubicin-induced apoptosis, induced DNA synthesis, and promoted cell cycle progression. The phenotype of the alphaMHC-survivin(-/-) mice also allowed us to determine the minimum cardiomyocyte number sufficient for normal cardiac function. In human cardiomyopathy, survivin was potently induced in the failing heart and downregulated again after hemodynamic support by a left ventricular assist device. Its expression positively correlated with the mean cardiomyocyte DNA content. CONCLUSIONS: We suggest that the ontogenetically determined cardiomyocyte number may be an independent factor in the susceptibility to cardiac diseases. Through its profound impact on both cardiomyocyte replication and apoptosis, survivin may emerge as a promising new target for myocardial regeneration.

High-density lipoproteins and their constituent, sphingosine-1-phosphate, directly protect the heart against ischemia/reperfusion injury in vivo via the S1P3 lysophospholipid receptor.

BACKGROUND: All treatments of acute myocardial infarction are aimed at rapid revascularization of the occluded vessel; however, no clinical strategies are currently available to protect the heart from ischemia/reperfusion injury after restitution of blood flow. We hypothesized that some of the cholesterol transport-independent biological properties of high-density lipoprotein (HDL) implied in atheroprotection may also be beneficial in settings of acute myocardial reperfusion injury. METHODS AND RESULTS: In an in vivo mouse model of myocardial ischemia/reperfusion, we observed that HDL and its sphingolipid component, sphingosine-1-phosphate (S1P), dramatically attenuated infarction size by approximately 20% and 40%, respectively. The underlying mechanism was an inhibition of inflammatory neutrophil recruitment and cardiomyocyte apoptosis in the infarcted area. In vitro, HDL and S1P potently suppressed leukocyte adhesion to activated endothelium under flow and protected rat neonatal cardiomyocytes against apoptosis. In vivo, HDL- and S1P-mediated cardioprotection was dependent on nitric oxide (NO) and the S1P3 lysophospholipid receptor, because it was abolished by pharmacological NO synthase inhibition and was completely absent in S1P3-deficient mice. CONCLUSIONS: Our data demonstrate that HDL and its constituent, S1P, acutely protect the heart against ischemia/reperfusion injury in vivo via an S1P3-mediated and NO-dependent pathway. A rapid therapeutic elevation of S1P-containing HDL plasma levels may be beneficial in patients at high risk of acute myocardial ischemia.

Immunomodulator FTY720 Induces eNOS-dependent arterial vasodilatation via the lysophospholipid receptor S1P3.

The novel immunomodulator FTY720 is effective in experimental models of transplantation and autoimmunity, and is currently undergoing Phase III clinical trials for prevention of kidney graft rejection. FTY720 is a structural analogue of sphingosine-1-phosphate (S1P) and activates several of the S1P receptors. We show that FTY720 induces endothelium-dependent arterial vasodilation in phenylephrine precontracted mouse aortae. Vasodilation did not occur in thoracic aortic rings from eNOS-deficient mice, implicating and effect dependent of activation of the eNOS/NO pathway. Accordingly, FTY720 induced NO release, Akt-dependent eNOS phosphorylation and activation in human endothelial cells. For biological efficacy, FTY720 required endogenous phosphorylation, since addition of the sphingosine kinase antagonist N',N-dimethylsphingosine (DMS) prevented activation of eNOS in vitro and inhibited vasodilation in isolated arteries. The endothelial phosphorylation of FTY720 was extremely rapid with almost complete conversion after 10 minutes as determined by mass spectrometry. Finally, we identified the lysophospholipid receptor S1P3 as the S1P receptor responsible for arterial vasodilation by FTY720, as the effect was completely abolished in arteries from S1P3-deficient mice. In summary, we have identified FTY720 as the first immunomodulator for prevention of organ graft rejection in clinical development that, in addition, positively affects the endothelium by stimulating NO production, and thus potentially displaying beneficial effects on transplant survival beyond classical T cell immunosuppression.

HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3.

HDL is a major atheroprotective factor, but the mechanisms underlying this effect are still obscure. HDL binding to scavenger receptor-BI has been shown to activate eNOS, although the responsible HDL entities and signaling pathways have remained enigmatic. Here we show that HDL stimulates NO release in human endothelial cells and induces vasodilation in isolated aortae via intracellular Ca2+ mobilization and Akt-mediated eNOS phosphorylation. The vasoactive effects of HDL could be mimicked by three lysophospholipids present in HDL: sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and lysosulfatide (LSF). All three elevated intracellular Ca2+ concentration and activated Akt and eNOS, which resulted in NO release and vasodilation. Deficiency of the lysophospholipid receptor S1P3 (also known as LPB3 and EDG3) abolished the vasodilatory effects of SPC, S1P, and LSF and reduced the effect of HDL by approximately 60%. In endothelial cells from S1P3-deficient mice, Akt phosphorylation and Ca2+ increase in response to HDL and lysophospholipids were severely reduced. In vivo, intra-arterial administration of HDL or lysophospholipids lowered mean arterial blood pressure in rats. In conclusion, we identify HDL as a carrier of bioactive lysophospholipids that regulate vascular tone via S1P3-mediated NO release. This mechanism may contribute to the vasoactive effect of HDL and represent a novel aspect of its antiatherogenic function.